In the search for new potential chemotherapeutics, the compounds’ toxicity to healthy cells is an important factor. The brain with its functional units, the neurons, is especially endangered during the radio- and chemotherapeutic treatment of brain tumors. The effect of the potential compounds not only on neuronal survival but also neuronal function needs to be taken into account. Therefore, in this study we aimed to comprehend the biological effects of chemotherapeutic xCT inhibition on healthy neuronal cells with our synaptic optogenetic function analysis tool (SOFA). We combined common approaches, such as investigation of morphological markers, neuronal function and cell metabolism. The glutamate-cystine exchanger xCT (SLC7A11, system Xc−) is the main glutamate exporter in malignant brain tumors and as such a relevant drug target for treating deadly glioblastomas (WHO grades III and IV). Recently, two small molecules termed sorafenib (Nexavar) and erastin have been found to efficiently block xCT function. We investigated neuronal morphology, metabolic secretome profiles, synaptic function and cell metabolism of primary hippocampal cultures (containing neurons and glial cells) treated with sorafenib and erastin in clinically relevant concentrations. We found that sorafenib severely damaged neurons already after 24 h of treatment. Noteworthy, also at a lower concentration, where no morphological damage or metabolic disturbance was monitored, sorafenib still interfered with synaptic and metabolic homeostasis. In contrast, erastin-treated neurons displayed mostly inconspicuous morphology and metabolic rates. Key parameters of proper neuronal function, such as synaptic vesicle pool sizes, were however disrupted following erastin application. In conclusion, our data revealed that while sorafenib and erastin effectively inhibited xCT function they also interfered with essential neuronal (synaptic) function. These findings highlight the particular importance of investigating the effects of potential neurooncological and general cancer chemotherapeutics also on healthy neuronal cells and their function as revealed by the SOFA tool.

Brain tumors are fast proliferating and destructive within the brain microenvironment. Effective chemotherapeutic strategies are currently lacking which combat this deadly disease curatively. The glioma-specific chloride ion channel represents a specific target for therapy. Chlorotoxin (CTX), a peptide derived from scorpion venom, has been shown to be specific and efficacious in blocking glioma Cl− channel activity. Here, we report on two new derivatives (termed CA4 and CTX-23) designed and generated on the basis of the peptide sequence alignments of CTX and BmKCT. The novel peptides CA4 and CTX-23 are both effective in reducing glioma cell proliferation. In addition, CTX, CA4 and CTX-23 impact on cell migration and spheroid migration. These effects are accompanied by diminished cell extensions and increased nuclear sizes. Furthermore, we found that CA4 and CTX-23 are selective with low toxicity against primary neurons and astrocytes. In the ex vivo VOGiM, which maintain the entire brain tumor microenvironment, both CTX and CA4 display anti-tumor activity and reduce tumor volume. Hence, CTX and CA4 reveal anti-angiogenic properties with endothelial and angiogenic hotspots disrupting activities. These data report on the identification of two novel CTX derivatives with multiple anti-glioma properties including anti-angiogenesis.

Hypersynchronous neuronal excitation manifests clinically as seizure (ictogenesis), and may recur spontaneously and repetitively after a variable latency period (epileptogenesis). Despite tremendous research efforts to describe molecular pathways and signatures of epileptogenesis, molecular pathomechanisms leading to chronic epilepsy remain to be clarified. We hypothesized that epigenetic modifications may form the basis for a cellular memory of epileptogenesis, and used a primary neuronal cell culture model of the rat hippocampus to study the translation of massive neuronal excitation into persisting changes of epigenetic signatures and pro-epileptogenic target gene expression. Increased spontaneous activation of cultured neurons was detected 3 and 7 days after stimulation with 10 μM glutamate when compared to sham-treated time-matched controls using calcium-imaging in vitro. Chromatin-immunoprecipitation experiments revealed short-term (3 h, 7 h, and 24 h) and long-term (3 d and 2 weeks) changes in histone modifications, which were directly linked to decreased expression of two selected epilepsy target genes, e.g. excitatory glutamate receptor genes Gria2 and Grin2a. Increased promoter methylation observed 4 weeks after glutamate stimulation at respective genes suggested long-term repression of Gria2 and Grin2a genes. Inhibition of glutamatergic activation or blocking the propagation of action potentials in cultured neurons rescued altered gene expression and regulatory epigenetic modifications. Our data support the concept of a cellular memory of epileptogenesis and persisting epigenetic modifications of epilepsy target genes, which are able to turn normal into pro-epileptic neurons and circuits.

Analyzing the connectivity of neuronal networks, based on functional brain imaging data, has yielded new insight into brain circuitry, bringing functional and effective networks into the focus of interest for understanding complex neurological and psychiatric disorders. However, the analysis of network changes, based on the activity of individual neurons, is hindered by the lack of suitable meaningful and reproducible methodologies. Here, we used calcium imaging, statistical spike time analysis and a powerful classification model to reconstruct effective networks of primary rat hippocampal neurons in vitro. This method enables the calculation of network parameters, such as propagation probability, path length, and clustering behavior through the measurement of synaptic activity at the single-cell level, thus providing a fuller understanding of how changes at single synapses translate to an entire population of neurons. We demonstrate that our methodology can detect the known effects of drug-induced neuronal inactivity and can be used to investigate the extensive rewiring processes affecting population-wide connectivity patterns after periods of induced neuronal inactivity.